Biosensors use biological elements to detect and quantify analytes and have applications in toxicology, pharmacology, medical diagnostics, and environmental monitoring. Biosensors that include living cells as sensing elements are referred to as cytosensors. Cytosensors are function-based sensors because they typically detect substances based on the effect of an analyte on functional activities of sensor cells. Sensor cells provide a large number of functional targets for detecting biologically active agents. These functional targets (such as enzymes, receptors, membranes, cytoskeletal structures, cellular organelles, second messenger signals, cytoplasmic elements and others) may be partly specified or unspecified at the time a measurement is performed and can remain unspecified even for a test standardized for a particular application. In addition, classes of analytes can be identified with or without identification of any specific analytes. For example, irritants in cosmetics can be detected by a standardized reaction with a cytosensor without knowledge of a reaction mechanism. Such a test need not identity any specific irritants but may indicate the presence of irritants. Such methods can serve as alternatives to animal testing in applications such as food and drug testing. Because cytosensors are based on living cells, cytosensor measurements can be configured to replace live animal tests.
The living cells used in cytosensors must be maintained for sufficiently long time periods for performance of cytosensor tests and cells that exhibit longevity and ruggedness after extraction are particularly useful. The cells must also be configured so that cell changes in response to analyte exposures can be detected.
Okun et al., U.S. Pat. No. 5,919,646, describes an apparatus and method for real-time measurement of cellular response of a test compound. Elving, U.S. Pat. No. 4,985,353, discloses detection of a toxin produced by Bordetella pertussis based on a color change in a fish scale. Lerner et al., U. S. Pat. Nos. 5,462,856 and 6,051,386 disclose methods for identifying chemicals that act as agonists for a G-protein coupled cell surface receptor. Danosky and McFadden, Biosensors & Bioelectronics 12:925-936 (1997), describes biosensors based on the chromatic activities of fish melanophores. Weaver, U.S. Pat. No. 4,401,755, discloses a process for encapsulating a biologically active compound with a material, allowing the material to gel, and then detecting a response.
Living cells can be used to identify or quantify bioactive agents in samples based on changes in, for example, cell morphology and/or physiology. Such changes can be directly detected or detected with the aid of instrumentation. For example, cells can be arranged so that visual inspection is adequate to identify the presence of bioactive agents, or, alternatively, an optical measurement system or other instrumentation can be provided.
In representative embodiments, methods of using at least one chromatophore to detect bioactive agents are provided. These methods involve placing the chromatophore in functional contact with a sample containing at least one suspected bioactive agent. Functional contact in this context means than the chromatophore is placed in a position relative to the sample such that the chromatophore can react to the various bioactive agents in the sample. Hence, the term has a broader meaning that physical contact.
In other representative examples, living cells used as sensors include chromatophores that exhibit changes in color, morphology, and/or distribution of pigment in response to selected analytes. These changes are monitored with or without additional instrumentation.
According to another aspect of the invention, encapsulated chromatophores are configured for the detection of bioactive agents. In a particular example, encapsulated chromatophores are at least partially surrounded in a coating configured to maintain the chromatophores in a suitable environment and in functional contact with test samples.
In further embodiments, cytosensors are configured to detect samples that include a variety of different types of compounds. For example, samples can contain organisms such as bacteria, fungi, viruses, plants, and animals. Samples can also contain neurotransmitters, hormones, intracellular signal transduction agents, pharmaceutically active agents, toxic agents, agricultural chemicals, chemical toxins, biological toxins, microbes, and animal cells such as neurons, liver cells, and immune system cells. Test kits for use in detecting bioactive agents are provided. In some examples, the test kits include a nutrient solution, at least one chromatophore, and a positive control solution.
Cytosensors are configured to expose chromatophores and to detect changes in the chromatophores. The cytosensors include a reaction chamber configured to retain chromatophores and an optical detection system situated to detect associated optical changes. A signal processing system detects chromatophore changes based on a red-green-blue or hue-saturation-value color representation of a transmitted or reflected optical flux that is received by the optical detection system. In other examples, such detection systems and methods are configured to detect changes in the coloration of cells other than chromatophores.
Cytosensors include a vessel defining an inlet configured to receive at least one bioactive material or test compound. A mixing zone is provided that is fluidically coupled to receive the chromatophores and the test compound from the inlet. The mixing zone is configured to expose the chromatophores to the test compound. The cytosensor can also include means for mixing the bioactive unit with the chromatophores, and a sensor for detecting changes in chromatophore cell properties (such as, for example, color or other optical properties, electrical properties such as capacitance or conductivity or other properties). The cytosensors described herein can also contain at least one signal processing system coupled to the sensor and at least one processor that converts a digital output from the signal processing system into an analytical result.
Fish chromatophores of varies species of fish can be used. Examples include freshwater zebrafish, south American cichlids, African cichlids, saltwater damsels, goldfish, gouramis, and other species. In addition, chromatophores can be based on fish scales, fin tissues, and the like. For use in cytosensors, encapsulation of chromatophores or analytes is unnecessary.
Cytosensors are configured so that chromatophores can detect agents that act on functional targets selected from a group consisting of enzymes, receptors, membranes, cytoskeletal structures, cellular organelles, second messenger signals, cytoplasmic elements and the like at a molecular and/or a cellular level. Such targets may be partly or wholly undefined at the time the measurement is performed. These targets-trigger a detectable response to an active compound and can remain undefined even when a test is commonly used or is standardized for a particular industrial, commercial, or academic application.
These and other features of the invention are set forth below with reference to the accompanying drawings.